Download Immunological Characterizations of Patients with Acquired Immune

[CANCER RESEARCH
44, 5894-5901,
December 1984]
Immunological Characterizations of Patients with Acquired Immune Deficiency
Syndrome, Acquired Immune Deficiency Syndrome-related Symptom
Complex, and a Related Life-Style1
Evan M. Hersh,2 P. W. A. Mansell, J. M. Reuben, A. Rios, and G. R. Newell
Departments of Clinical Immunology and Biological Therapy [E. M. H., P. W. A. M., J. M. R., A. R.], Laboratory Medicine [E. M. H., J. M. R.], and Cancer Prevention
[P. W. A. M., A. R., G. R. N.], The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030
ABSTRACT
Immunological, hematological, and biochemical studies were
done at the time of referral in 135 homosexual subjects, 28 of
whom were symptom free (SF), 74 of whom had the acquired
immune deficiency syndrome (AIDS)-related symptom complex
(ARC), and 33 of whom had AIDS with Kaposi's sarcoma,
opportunistic infection, or both. Of 38 laboratory parameters, 11
were significantly different than controls in the SF patients, 19 in
the ARC patients, and 20 in the AIDS patients. In SF patients,
delayed hypersensitivity was significantly suppressed for 6 of 12
recall antigens. In addition, the percentage of circulating lympho
cytes, the percentage of T3+ cells, the percentage and absolute
number of T4+ cells, the T4/T8 ratio, the blastogenic responses
to phytohemagglutinin, pokeweed mitogen, and concanavalin A
were depressed significantly in this group. In contrast, the per
centage and absolute granulocyte count, the serum lysozyme,
and the serum thymosin <*i were significantly elevated in these
patients. In patients with more advanced disease (ARC and
AIDS), immunological and hematological parameters tended to
worsen. Thus, in the AIDS patients the white blood cell count,
percentage, and absolute T11+ cells, absolute T3+ cells, per
centage of T4+ cells and absolute level of B-cells, as well as the
monocyte adherence and delayed hypersensitivity responses to
12 of 12 recall antigens were depressed. Serum levels of thy
mosin «! were equally elevated in all three groups. Serum
Interferon was found in 15 of 18 opportunistic infection patients
with or without Kaposi's sarcoma, in 3 of 9 Kaposi's sarcoma
patients without opportunistic infection, but in none of the ARC
or SF patients. This study has demonstrated that SF sexually
active homosexuals have a characteristic pattern of immune
deficiency and that immunodeficiency worsens as one compares
SF to ARC to AIDS patients. The study has provided a data
base for the development of prognostic criteria and for charac
terization and evaluation of immunorestorative and immunomodulatory therapy.
since its initial identification in 1979 (1). Initially detected in
homosexuals in New York City and California, it has now been
identified in at least 5 other high-risk groups (32), is occasionally
seen in subjects without a known predisposing life-style factor,
and it has spread to countries in both Europe (11) and the Far
East. There is no known treatment for the underlying immuno
deficiency, and it is associated with a mortality of at least 40%
and perhaps as high as 80% or more (10, 27).
AIDS is a clinical syndrome characterized by major Ol or
cancer, mainly KS, or both, associated with at least 2 defined
immune abnormalities in patients without a known cause for the
immunodeficiency. Known causes of immunodeficiency which
exclude patients from diagnosis of AIDS include ¡mmunosuppressive drug therapy, preexisting cancer or other known dis
eases with secondary immunodeficiency, and known congenital
or acquired immunodeficiency diseases.
Groups in which AIDS has been observed to occur include
homosexuals (13), Haitian immigrants (2), hemophiliacs (3), i.v.
drug abusers (12), prisoners (14), offspring of one of the above
(31), and heterosexual partners of one of the above (28). Others
may be discovered, since about 5% of cases have no recognized
predisposing factor.
A complex of symptoms (fever, night sweats, lymphadenopathy, weight loss, diarrhea, and fatigue) usually precedes the
development of AIDS and is now recognized as existing as an
entity itself, not necessarily progressing to the full-blown disease.
The presence of 2 or more of these for at least 3 months,
associated with a life-style risk factor, constitutes what has
variously been referred to as early or pre-AIDS, AIDS prodrome,
forme fruste of AIDS, or the ARC. It may or may not progress to
AIDS. We accept this definition if the patients also have 2 or
more defined immune abnormalities without another known
cause for immunodeficiency. The immune tests used to define
immune abnormalities include delayed hypersensitivity to recall
antigens, absolute T-cell count, T-helper cell count, helper/sup
pressor ratio, lymphocyte proliferative response to mitogens,
and natural killer cell activity (see "Materials and Methods"). We
INTRODUCTION
AIDS3 has become a major health problem in the United States
1Supported by Grants CA-34674 and CA-31544 from the National Cancer
Institute, and by grants from the President's Development Fund of M. D. Anderson
Hospital and the Kaposi's Sarcoma Foundation of Houston.
2 To whom requests for reprints should be addressed, at Clinical Immunology,
Box 41, M. D. Anderson Hospital and Tumor Institute, Houston, TX 77030.
3 The abbreviations used are: AIDS, acquired immune deficiency syndrome;
ARC, acquired immune deficiency syndrome-related system complex; BUN, bloodurea-nitrogen; DTH, delayed-type hypersensitivity; KS, Kaposi's sarcoma; Ol, op
portunistic infection; PHA, phytohemagglutinin; PWM, pokeweed
symptom-free.
Received May 31, 1984; accepted September 4, 1984.
CANCER
mitogen; SF,
RESEARCH
only require that these be present once.
We have hypothesized that the following sequence of events
may occur in the development of this syndrome. First, there is
the presence of a life-style or group of risk factors which predis
pose to some degree of immune deficiency. Then the patient
becomes infected with the presumed transmissible causative
agent. Following this, there are manifestations of a prodromal
phase and, finally, the fully expressed disease entity with Ol, KS,
or both. We felt that immunological characterization of patients
at the beginning of this sequence might be useful. It might provide
clues to early detection, mechanism of disease pathogenesis,
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IMMUNOLOGICAL
CHARACTERIZATION
and identifiable factors to be used as diagnostic, prognostic, and
therapeutic targets. Such a study was made possible by the
establishment of an AIDS clinic in the Department of Cancer
Prevention at M. D. Anderson Hospital. Preliminary evaluation of
the first 33 cases (30) showed that a defined complex of immune
abnormalities was detectable in the "early" patients as well as
those with KS and/or Ol. Complete immune evaluation has now
been carried out in the first 135 of these cases and is reported
herein. The study confirms a defined complex of immune abnor
malities which is present in the asymptomatic high-risk patients
and is most severe in those with KS or Ol. Relationships among
certain factors were found and will be described. The data
provide a useful set of criteria for diagnostic, prognostic, and
therapeutic studies and prove that defined immune abnormalities
precede the development of AIDS.
MATERIALS AND METHODS
All patients were referred to one of us by their physician from January
3,1982, to October 1,1983, because either AIDS or ARC was suspected
or because the patient, knowing the relationship between AIDS and the
homosexual life-style, asked for referral. Evaluation included a complete
history, physical examination, a detailed life-style questionnaire, and
standard medical as well as extensive immunological tests. All proce
dures were approved by both the Institutional Research and Protection
of Human Subjects (Surveillance) Committees. All subjects gave their
informed consent for the evaluation, and all clinical and laboratory data
were maintained in confidence.
Of the 135 male subjects, all were sexually active homosexuals, 28
were asymptomatic (also referred to in this paper as SF), 74 had 2 or
more of the AIDS-related symptoms (fever, night sweats, lymphadenopathy, diarrhea, weight loss, and fatigue) without cancer or Ol (referred to
as ARC), and 33 had either KS, Ol, or both. These subjects averaged
34 years of age and averaged 2.4 sexual partners/week; 51.6% practiced
anal intercourse as active participants and 67.7% as passive participants;
19.4% practiced active fisting and 12.8% passive fisting; 58.1% used
marijuana, 71.0% nitrites, and 15.0% other recreational drugs. Prior
syphillis was noted in 67%, prior gonorrhea in 79%, and prior hepatitis
in 54%. The distribution of life-style factors and associated diseases
among the 3 groups and their relationship to AIDS will be reported
separately.
Conventional medical tests included complete blood count; differential
and platelet count; absolute granulocytes, lymphocytes, and monocytes;
and SMA 1260 which includes total protein, albumin, globulin, calcium,
phosphorus, BUN, creatinine, bilirubin, alkaline phosphatase, lactic dehydrogenase, and serum glutamic-oxaloacetic acid transaminase. Other
tests included serum protein electrophoresis, quantitative immunoglobulins, and measurement of serum antibody titers to Epstein-Barr virus
cytomegalovirus, and hepatitis viruses.
Immunological tests carried out are listed below. All have been de
scribed previously by our laboratory in the literature (30). They include:
DTH skin testing by intradermal injection of the recall antigens derrnatophytin, Candida, streptokinase-streptodomase(Varidase),
mumps, and
purified protein derivation (30); and DTH skin testing with the new
Multitest Skin Test Device (Merieux Institute) which delivers antigens via
8 tine multipuncture heads (22). This device administers trichophytin,
Candida, tetanus, diphtheria, Streptococcus, Proteus, and tuberculin
antigens as well as a glycerol control with a single stroke. Peripheral
blood lymphocytes were evaluated for the percentage and absolute
number of E-rosette-forming cells (cell-forming rosettes with neuraminidase-treated sheep RBC), cells with the surface antigens T11, T3, T4,
and T8, and cells with the surface ¡mmunoglobulins IgM and IgD (17).
These were determined using the Ortho-mune monoclonal antibodies
and with Fab'2 polyclonal antibody to IgM and IgD from Cappel Labora
tories. Since OKT11 recognizes the E-rosette receptor and since we
have found that the percentage of cells positive for cell-forming rosettes
with neuraminidase-treated sheep RBC and OKT11 were identical, they
are used interchangeably. The percentage and number of esterase plus
monocytes were determined by a nonspecific esterase stain. Peripheral
blood lymphocyte proliferative responses to PHA, concanavalin A, and
PWM were evaluated by tritiated thymidine incorporation after 3 days in
culture (25), natural killer cell cytotoxicity (33), and antibody-dependent
cell-mediated cytotoxicity (18) to human RBC and the CEM T-leukemia
cell line were determined. Monocyte adherence (the numbers of mono
cytes which adhere to plastic and transform to macrophages in 7 days)
was also determined (15). Serum lysozyme was measured using the
Worthington Biochemicals kit. Dr. Allen Goldstein (George Washington
University) measured the serum thymosin «i by a radioimmunoassay
(26, 29), and Dr. Olivia Prebble (Uniformed Services Medical Center)
measured the serum level of a-interferon (6).
Control subjects consisted of heterosexual male and female hospital
employees, faculty, and medical or graduate students in the same age
range as the patients. All controls were evaluated concurrent with the
patient groups during the 9 months during which the study was con
ducted. There were 192 subjects evaluated for WBC and differential and
for leukocyte surface markers. There were 58 subjects evaluated for
lymphocyte blastogenic responses. Forty subjects were evaluated for
cell-mediated cytotoxicity, 37 for monocyte adherence, 47 for serum
lysozyme, and 50 for thymosin «,.For serum a-interferon, controls were
not run, since this activity is found in less than 3% of the sera of healthy
normals. Delayed-type hypersensitivity to recall antigens was evaluated
in 40 normal subjects.
Two well-established statistical packages were used via the Cyber
600 main-frame computer of the University of Texas Health Science
Center at Houston, SPSS and MINITAB. Distribution of patients and
normals according to arbitrary points of discrimination for each laboratory
value were evaluated by the x2 test. Through this, the 3 patient groups
were compared to each other, the 3 groups were compared individually
to the controls, and all 135 patients were compared to controls. In
addition, the frequency distributions of the groups were compared by
the Anova and Kruskal-Wallis one-way analysis of variance for nonparametric values. The relationship of patient clinical and immunological
values to each other were tested by the Spearman Rank correlation test.
For the latter, because of the large number of variables under study,
only r values with p values sO.01 and r values with p values between
0.01 and 0.03 were considered significant and are reported in this paper.
RESULTS
A comparison of the 31 relevant hematological and immuno
logical values of all 135 patients with those of the normals is
given in Table 1. Chemical (SMA 1260) values are not shown,
since they were essentially normal in the patients. Also given are
the percentage of patients whose values were completely out
side of the normal range for our laboratories or greater than 2
S.D.s from the normal mean. The 135 patients, as a group,
showed significantly increased percentages of and absolute val
ues for granulocytes and significantly reduced percentages of
and absolute values for lymphocytes compared to the normal
controls. While not significantly reduced, the hemoglobin and
hematocrit values were below the normal range in about 20% of
the patients, while the platelets were below the normal range in
only 8% of the cases. These data indicate a potential important
hematological abnormality in patients with AIDS, ARC, and the
symptom-free status.
OKT11, OKT3, OKT4, and OKT8 from Ortho Diagnostics, Raritan, NJ,
CANCER
RESEARCH
OF AIDS
Evaluation of the cell surface markers on peripheral blood
lymphocytes revealed that the percentage of E-rosette-forming
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IMMUNOLOGICAL
CHARACTERIZATION
OF AIDS
Table 1
Mean host defense and related parameters in patients and normals
Serum levels of thymosin a, were elevated in virtually every
patient in whom it was measured and was highly significantly
of abnor
abnormal compared to the controls. The monocyte adherence
malpatients16.045.020.550.022.630.811.124.016.08.033.633.040.842.068.552.061.610.063.837.85.016.03.05
of the patients was depressed compared to normal (p < 0.01).
Parameters for which at least 50% of the patients were
abnormal included percentage of lymphocytes, percentage of
and absolute T4+ cells, percentage of T8 cells, T4/T8 ratio,
monocyte adherence, and serum thymosin a, level.
Table 2A outlines the skin test reactivity to the conventional
skin test battery of the total patient group compared to normal.
Both the response score which converts the skin reaction sizes
to an arbitrary scale and sums them, and the number of positive
skin tests were significantly lower in the patients compared to
the normals. Among the individual skin tests evaluated, only the
mumps reaction was significantly reduced in the patients com
pared to the controls. Forty % of the patients were completely
anergic, while an additional 16% had only one positive test.
Table 2B shows similar data on DTH using the multitest skin
test battery. This battery was more sensitive than the conven
tional battery in detecting the immunodeficiency associated with
ParameterWBC(cummx
10")%
PMNAbsolute
of
(percu PMN
103)%
mm x
lymphocyteAbsolute
of
lymphocytes%
monocylesAbsolute
of
monocytesHgb(g/dl)Hct
(%)Platelets
mmX103)%
(per cu
E-rosettes(T11+)Absolute
of
(percu T11-1O3)%mm x 1
T3+Absolute
of
T3+%
T4+Absolute
of
T4+%
T8+Absolute
of
T8+T4/T8%
M+D+Absolute
of
M+D+ADCC-HRBC
Table 2
Delayedhypersensitivity responses
Skin test diameters are mean values for mm induration. Values shown are 48hr readings.
ofTC
(%
lysis)ADCC-CEM
ofTC
(%
lysis)NK-K562
Parameter
TClysis)Cont
(% of
Patients
Normals
A. Responsesto the conventionalskin test battery
Response score
8.4
11.7
No. of positive/5
1.3
2.2
Dermatophytin(diameter)
3.4
3.3
Candida (diameter)
11.8
11.5
Streptococcus (diameter)
9.4
12.8
Mumps (diameter)
9.1
15.4
PPD (diameter)
2.8
2.0
blastogenesis(cpmx
103)PHA
blastogenesisPWM
blastogenesiscon-A
blastogenesisMonocyte
adherenceSerum
p value
<0.01
<0.01
NS"
NS
NS
0.05
NS
B. Responsesto the multitest battery
No. of positive/7
1.5
4.1
<0.01
Tricomonas(diameter)
0.6
1.7
<0.01
a,dig/ml)Patients6.163.94.328.82.15.30.3514.643.7263.268.11.462.11.324.40.5736.10.720.8214.00.2710.151.914.91.080.034.737.69.111.62430Normals6.559.13.936.32.34.30.2814.0-18.040.0-54.
thymosin
Candida (diameter)
2.3
4.1
<0.01
Streptococcus (diameter)
1.1
2.9
<0.01
" NS, not significant; PMN, polymorphonuclear neutrophilic leukocytes; Hgb,
Diphtheria(diameter)
1.2
2.9
<0.01
hemoglobin; Hct, hematocrit; ADCC. antibody-dependent cellular cytotoxicity;
Tetanus (diameter)
2.6
5.3
<0.01
HRBC, human red blood cells; Cont, controls; con A, concanavalin A; NK, natural
Proteus (diameter)
1.1
2.3
<0.01
killer.
Tuberculin (diameter)
0.9
2.4
<0.01
* NS, not significant; PPD, purified protein derivation.
lysozyme(eg/ml)Serum
cells was normal, while the absolute level was significantly re
duced. Levels (both relative and absolute) of OKT3- and OKT4positive cells were significantly reduced, while the percentage of
OKT8-positive cells was significantly increased. In contrast, the
absolute level of OKT8+ cells was in the normal range. The ratio
of helper (T4+) to suppressor (T8+) cells was inverted, being
0.85 in the patients and 1.85 in the controls. The percentages
and absolute values for B-cells (surface ¡mmunoglobulin M+0
positive) were normal in the patient.
Evaluation of cell-mediated cytotoxicity (natural killer and an
tibody-dependent cellular cytotoxicity) showed no differences
between patients and controls. The lymphocyte blastogenic re
sponses of the patients were markedly depressed compared to
the normals (p < 0.01 for each of the 3 mitogens). The thymidine
incorporation of the unstimulated lymphocytes was the same in
the patients and normals. Serum lysozyme was significantly
elevated in the patient (p < 0.01). No patient had impaired renal
function or an elevated BUN or creatinine which can explain
elevated lysozyme, since this enzyme is excreted in the urine.
CANCER
RESEARCH
Tabled
Serum thymosin a, levels
All patient groups were significantly higher than control; p < 0.01.
Thymosin a, (pg/ml)
GroupSymptom-free
S.D.2387
±
ARC
AIDS
ControlMean
±698
990-4050
2479 ±725
2516
1191-4590
2413 ±655
2357
272-702
456 ±138Median2260
416Range1130-3687
Table 4
Circulating interferon in symptom-free, ARC, and AIDS patients
Data shown are x2 between all groups using a 2 x 4 table; p < 0.001.
Classification of disease
Serum
interferonPresent + ARC0
AbsentTotalSF
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O
+ I7
69Ol9
110KS
18Total19
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7493
IMMUNOLOGICAL
CHARACTERIZATION
OF AIDS
Tables
Mean host defense parameters in 3 patient groups compared to normals
Asymptomatic
Symptomatic
Parameter
Value
WBC
103)%(per cu mm x
PMNAbsolute
of
103)% PMN (per cu mm x
lymphocytesAbsolute
of
lymphocytes%
monocytesAbsolute
of
monocytesHgb(g/dl)HctPlatelets%ofE-rosettes(T11+)Absolute
KS/OI
Value
Value
Normal value
03)%
T11+ (per cu mm x 1
T3+Absolute
of
T3+%ofT4+Absolute
T4+%
T8Absolute
of
T8+T4/T8%
M+D+Absolute
of
M+D+ADCC-HRBC
lysis)ADCC-CEM
(% of TC
lysis)NK-K562(% of TC
lysis)Cont (% of TC
103)PHA
LBR (com x
LBRPWM
LBRcon-A
LBR7.067.64.926.11.74.20.3415.043.028666.81.559.91.328.70.6429.60.621.1114.10.2411.750.714.51.076.440.936.7NS"<0.010.02<0.01NSNSNSNSNSNSNSNS0.02NS<0.01<0.01NSNS<0.01N
Monocyte adherence
12.6
9.1
NS
9.1
NS
13.2
Lysozyme (jig/ml)
<0.01
2387
2479
Thymosin a, (ng/ml)
8 All p values compare patient groups to normal controls.
0 NS, not significant; LBR, low Wastogenicresponse. For other abbreviations, see Table 1.
AIDS. In addition to a significant difference in the number of
positive tests per 7, the individual tests were each significantly
lower in the patients compared to the controls.
Table 3 outlines the serum thymosin <*i values. There was
evaluation of thymosin «,in almost all of the patients. This value
was elevated regardless of the clinical status and was as ele
vated in the asymptomatic subjects as in those with ARC and
AIDS. There was almost no overlap with the controls. Control
values on Houston normals and on Washington, DC normals
were comparable.4
Table 4 gives the relevant data on the serum a-interferon in
the patients. Of the 93 patient sera evaluated, only 19 were
found to be positive. All of these positive sera were from patients
with either KS, Ol, or both. None of the asymptomatic or ARC
patients had detectable serum interferon levels. It is of interest
that virtually all of the Ol patients with or without KS were
positive (15 of 18), while only 3 of 9 KS patients who did not
have Ol showed this finding.
Table 5 compares the 3 patient groups (SF, ARC, and AIDS)
individually to the normals. The mean values for each group as
well as the degrees of significance, determined by the KruskalWallis one-way analysis of variance, are given. Eleven of 31
parameters were significantly different than the controls among
the asymptomatic subjects, 19 among the symptomatic subjects,
and 19 among those with KS and/or Ol. If one also includes the
<0.01
4.8
<0.01
15.9
<0.01
<0.01
10.5
2412
<0.01
<0.01
7.36
456
presence of circulating interferon, then 20 parameters were
abnormal in the latter group. The relative and absolute levels of
granulocytes were increased in the SF patients, suggesting both
infection and the capacity of the marrow to respond. Relevant
values which were not reduced in the asymptomatic group
included the absolute lymphocyte count, relative and absolute
levels of E-rosette-forming cells, serum lysozyme, and monocyte
adherence. These values were significantly abnormal compared
to the controls in one or both of the 2 other groups. These
patients showed depressed or abnormal percentage of T3, per
centage of and absolute T4-positive cells, a significantly de
pressed T4/T8 ratio, low blastogenic responses, and an elevated
thymosin at level. Blood chemistries were normal in all 3 patient
groups. In general, immunological abnormalities became more
severe as one moved from SF to ARC to AIDS for each of the
parameters for which abnormalities were found.
Table 6 compares the skin test reactivity of the 3 groups of
patients with each other and the controls. In general, skin test
reactivity declined in the AIDS compared to ARC and ARC
compared to SF patients. The AIDS patients had very little skin
test reactivity. The reactions to the multitest battery were much
more sensitive than to the conventional battery in detection of
immune deficiency in these patients. The 3 groups differed from
each other significantly for each skin test parameter by x2 test
Table 7 shows the evaluation of the differences between the
3 major patient groups. The groups were compared for each
4A. Goldstein, personal communication.
CANCER
RESEARCH
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1984
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IMMUNOLOGICAL
CHARACTERIZATION
OF AIDS
Tables
Skin test reactivity at 48 hr
Skin test diameters are mean values for mm induration. Values shown are 48-hr readings.
AsymptomaticParameterDiameterPSymptomaticDiameterPKS/OIDiameterPNormal
diameterA.
batteryResponse
Reactivity at 48 hr to conventional
scoreNo.
tests/5DermatophytinCandidaStreptococcusPPDMumps8.71.83.018.19.51.39.7<0.01NS"NSNSNSNSNS7.61.41.39.89.21.48.1<0.010.02NSNSNSNSNS5.80.61.67.36.33.
of positive
batteryNo.
Reactivity at 48 hr to multitest
tests/7TricomonasCandidaStreptococcusDiphtheriaTetanusProteusTuberculin1.80.41.91.40.92.10.91.8<0.01<0.01<0.010.05<0.01<0.01<0.01NS1.80.41.90.81.12.00.90.9
of positive
"NS, not significant; PPD, purified protein derivative.
Table?
groupsParameterWBC%
Comparison of host defense parameters between 3 patient
lations of parameters which would be expected because of their
normal relatedness such as WBC count, and percentage and
absolute granulocyte count. It is of interest that the WBC count,
its components, and the T-cell parameters tended to move
together. For example, WBC count, T4 level, and T8 levels
PMNAbsolute
of
correlated directly with each other. This meant that the inverted
PMN%
helper/suppressor ratio was related to a fall in both cell types
lymphocytesAbsolute
of
lymphocytes%
but a greater fall in helper cells. Another observation of interest
monocytesAbsolute
of
was that the serum lysozyme level correlated inversely with
monocytesHgbHctPlateletsE-rosettesAbsolute
several of the hematological and immunological parameters.
Finally, because anecdotal observations suggested lack of
expression of certain markers on the lymphocytes of AIDS
patients, we evaluated the percentage of T11 cells failing to
E-rosettes%
mark with T3 and the percentage of T11 or T3+ cells failing to
T3+Absolute
of
mark
for T4 and T8. This was done by calculating the means,
T3+%
T4+Absolute
of
medians, etc., of T11 minus T3, T11 minus (T4+T8), and T3
T4+%
minus (T4+T8) for each of the groups. The medians of these
T8+Absolute
of
increased in the AIDS cases (Table 10). This was also true when
T8+T4/T8%
the data were evaluated by calculating the number of cases
M+D+Absolute
of
showing either T11 or T3 minus (T4+T8) exceeding 10%.
M+D+ADCC-HRBCADCC-CEMANOVA0.03NSNSNS0.05NSNS<0.01<0.01NSNSNSNS0.050.020.04<0.01NS<0.01NSNSNSNSPx2testNS"NS0.040.020.01NSNS<0.01<
DISCUSSION
'' NS, not significant. For other abbreviations, see Table 1.
parameter by the analysis of variance and the x2 test. The general
trend was for the degree of abnormality to be worse in more
advanced patients. This was significant for 20 of the 38 evaluated
values. It was of interest that this was not seen with the relative
or absolute level of E-rosette-forming cells and with the blastogenic responses to PHA and PWM. This was also the case for
the thymosin «!levels.
Table 8 gives the significant correlations at the p < 0.01 level,
and Table 9 gives the same at the p > 0.01 to 0.03 level between
the various study parameters in the patients. It is of interest that
several of the hematological and liver function parameters cor
related with the most relevant immunological values. The immune
parameters were lowest where the hematological and liver func
tion values were the lowest. Not shown are the obvious corre
CANCER
RESEARCH
These studies confirm and extend the observations by us (30)
and others (24, 31) on the immune and related functions of
patients with AIDS and the clinical states considered to be
associated with the subsequent development of AIDS.
Asymptomatic homosexuals, referred because they were con
cerned that their life-style (multiple sexual partners, multiple
sexually transmitted diseases) might eventuate in AIDS, were
found as a group to have a definable immunodeficiency consist
ent with that seen in a more severe form in AIDS. Low relative
levels of OKT3- and OKT4-positive lymphocytes, low absolute
levels of OKT4-positive lymphocytes, and inverted T4/T8 ratio,
impaired lymphocyte proliferative responses to PHA, PWM, and
concanavalin A and impaired skin test reactivity to the multitest
antigen battery were characteristic of this group. In addition,
they showed significantly elevated serum levels of thymosin at.
This confirms that a definable immunodeficiency is present in the
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1984
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IMMUNOLOGICAL
CHARACTERIZATION
high-risk group without evident symptoms or physical signs of
Tablee
Spearmanrank correlations with p < 0.01
pairHgb'-albuminHgb-%ofT4+Hgb-%
Variable
T8+Hgb-T4/T8WBC-absolute
of
ENWBC-absolute
T3WBC-absolute
T4WBC-absolute
T8WBC-monocyte
adherence%
of lymphocytes-abso
EN%
lute
of lymphocytes-abso
T3%
lute
of lymphocytes-abso
T4%
lute
of lymphocytes-abso
T8Absolute
lute
disease. It will be important to follow these SF patients serially
pair%ofT3-absoluteEN1>%
to determine if any of those with the most severe abnormalities
develop the symptomatic state (ARC) or AIDS characterized by
T4%ofT3-absoluteT8%
of T3-absolute
KS, Ol, or both. It will also be important to follow them to see if
their findings are stable or if they fluctuate with time. Finally, it
T3-absoluteM+DAbsolute
of
will be important to relate these findings to the presence of
antibody to HTLV-III and to culture evidence for those viruses
T3-absoluteT4Absolute
known to modulate immune response such as cytomegalovirus
T3-absoluteT8Absolute
and Epstein-Barr virus.
In all 3 patient groups, we found that virtually all patients had
T3-absoluteM+DAbsolute
an elevated serum thymosin at level. The etiology of this eleva
T3-T4/T8%ofT4-absoluteEN%ofT4-absoluteT3%
tion is obscure. We postulated previously end-organ failure with
a loss of feedback regulation as the cause (21). However,
Dardenne ef a/. (5) have recently demonstrated a low facteur
thymique serique level in AIDS patients by a functional assay.
Other possible causes include leakage from a damaged thymus
or ectopie production. The functional role of the thymus and its
hormones in AIDS needs to be elucidated for 2 reasons: (a) a
unique form of thymic involution has been described in AIDS (7);
and (b) thymic hormones are available for therapy of the syn
drome (25) if a suitable rationale can be established. At any rate,
whatever is responsible for the etiology of the syndrome, it must
act early on thymosin a, levels, since these are abnormal in the
asymptomatic group. While our controls were normals, other
investigators have found thymosin o, to be normal in hospitalized
cancer patients and in patients with infections.4
T8%
of T4-absolute
T4-absoluteM+D%ofT4-T4fT8Absolute
of
lymphocytes-absolute
ENAbsolute
lymphocytes-absolute
T3Absolute
lymphocytes-absolute
T4Absolute
lymphocytesT8%
absolute
T4-absoluteT8Absolute
T4-absoluteM+DAbsolute
T4-T4/T8%
T3%ofT8-T4fT8Lysozyme-%
of T8-absolute
T3%
of EN-absolute
T4%
of EN-absolute
T8Absolute
of EN-absolute
OF AIDS
ENLysozyme-%of
T3Lysozyme-%of
T4Albumin-% of
In contrast to the above, elevated serum interferon levels
T3AbsoluteEN-absolute
T4Albumin-absoluteM+DCalcium-absolute
of
T4AbsoluteEN-absolute
found only in the patients with KS, Ol, or both. In fact, only
EN-absolute T8r0.490.25-0.260.250.310.370.290.280.330.630.490.400.480.890.800.700.790.440.440.410.790.700.80Variable
were
in Ol
were detectable levels found in the majority of patients. This
suggests that it is a late event, is not related to etiology, and
lym
therefore is a response to the disease status, and it is an
phocytesCalcium-absolute
ENCalcium-absolute
associated phenomenon rather than a causative factor. How
T3Calcium-absolute
ever, its elevation has been proposed as a diagnostic test in the
T4Cateium-T4/T8Cateium-con-ACalcJum-monocyteadherenceLDH-T4/T8SGOT-ADCC-CEMSGOT-thymosin
disease (8).
Host defense parameter abnormalities were found in signifi
cantly greater frequency and at a significantly greater degree of
severity in the "prodromal" patients and to an even greater
degree in the patients with KS and/or Ol compared to the
symptom-free patients. This suggests either that the more ¡m-
a,r0.350.490.470.490.820.810.600.270.380.570.270.440.770.590.560.670.28-0.54-0.37-0.28-0.260.250.450.310.290.280.320.270.310.41-0.
munologically impaired patients are the ones that progress to
* For abbreviations, see Table 1.
6 EN, E-rosette-forming cells; SCOT, serum glutamic-oxaloacetic
acid transam-
inase.
Table 9
Spearman rank correlations with p values > 0.01 and < 0.03
Variable pair
r
% of lymprtocytes-monocyte adherence
% of ErAabsolute lymphocytes
PHA B1-T4/T8
Lysozyme-absolute EN
Lysozyme-absolute T4
Lysozyme-T4/T8
Albumin-absolute T3
Albumin-absolute T4
Albumin-T4/T8
Albumin-con-A
Calcium-% of T8
Calcium-PWM B1
LDH-absotute T4
8 EN, E-rosette-forming cells. For other abbreviations, see Table
CANCER
0.29
0.27
0.27
-0.27
-0.26
-0.20
0.25
0.24
0.20
0.26
-0.20
0.28
-0.22
1.
RESEARCH
AIDS or that, when AIDS occurs, it further suppresses the host
defense mechanisms. Serial follow-up studies should elucidate
this point. A combined effect is hypothesized, since many of the
AIDS-associated infections are known to suppress cellular im
munity.
Other investigators have reported an absolute increase in T8+
suppressor cells in ARC patients (9). This was not found in any
of our patient groups. The reason for this is unclear at present.
Another interesting aspect of our subset data is that T4+T8 did
not add up to T11 or T3 in the AIDS patients, suggesting an
increase in "null" cells. This would be consistent with diminished
mature and increased immature lymphocytes in the blood of
these patients. The concurrent low monocyte adherence and
high serum lysozyme deserve special comment. We speculate
that the peripheral blood is depleted by the need for macro
phages in infected tissues which results in redistribution of these
cells. Since they become activated by AIDS and/or its associated
infection, the serum lysozyme becomes elevated. The highest
values were observed in the middle patient group (ARC), sug-
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1984
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IMMUNOLOGICAL
CHARACTERIZATION
OF AIDS
TabtelO
Defects in cell surface marker expression in AIDS and related disorders
x* is used for T4+T8 - EN" normal versus ARC + AIDS,p = 0.03 and T4+T8 - T3 normal versus ARC +
AIDS,p = 0.04.
Parameter(% of positive cells)
Groups
EN-T3
(median)
EN-T4+T8
(median)
T3 - T4+T8
(median)
No. of cases
showing T4+T8
-EN*10%(%)
NormalSymptom-freeARCAIDS-3.33.01.83.5-1.14.04.612.01.13.32.61.56 (5)1
(4.5)9(13.2)4
No. of cases
showing T4+T8
-T3s10%(%)
(4.2)2
(9.0)6
(8.8)5(16.1)
(12.9)5
"EN, E-rosette-forming cells.
gesting that this group can be maximally activated after which
there may be a failure of the bone marrow to provide enough
cells for further activation. The high serum lysozyme was not
due to a renal failure, since all BUN and creatinine values were
normal in these patients.
The correlations between hematological and chemical values
on the one hand and immunological values on the other are also
worthy of special note. In general, the hemoglobin and WBC
count values and the albumin and calcium values correlated
directly and significantly with such immune parameters as the
level of T4+ cells and the helper/suppressor ratio. This means
that high values of one correlated with high values of the other.
The correlation of the T4+ cells and particularly the T4/T8 ratio
with functional immune parameters such as lymphocyte prolif
eration and DTH suggests a possible causitive relationship. We
have demonstrated previously that these patients have suppres
sor cell activity and it correlates with their helper/suppressor
ratio (16). The correlation between the hematological and im
mune parameters may be on the same basis, namely, suppressor
cell activity in the marrow. Immune regulation in normal marrow
does occur (23) and has been suggested as playing a role in this
disease (24).
The concurrent low monocyte adherence and high serum
lysozyme deserve special comment. We speculate that the pe
ripheral blood is depleted by the need for macrophages in
infected tissues. We suggest that this and the significant decline
in blood esterase-positive cells are due to a predominance of
tissue localization and activation of macrophages, a change in
monocyte kinetics and, late in the disease (in patients with KS
or Ol), a diminished marrow production of monocytes. Elevated
serum lysozyme is seen in both animals (4) and humans (19)
after administration of macrophage activators, and abnormally
low levels are noted in disorders of monocyte deficiency such as
hairy cell leukemia (20). It may also be that the activation of
monocytes results in monocyte-mediated suppression of im
mune responses.
In this study, we have evaluated hematological, chemical, and
immunological parameters in patients with AIDS, in its "prod
rome," and in symptom-free subjects with a high-risk homosex
ual life-style. A number of parameters were significantly abnormal
in asymptomatic subjects who belong to the high-risk group and
worsen significantly in the other 2 groups. Several parameters
(serum thymosin a1f PHA, and PWM responses, certain skin
tests) are equally abnormal in all 3 groups. About 20 parameters
are abnormal in the ARC and AIDS patients. One parameter
(serum interferon) is abnormal only in the KS and Ol patients.
These results have defined the spectrum of disease quantita
tively and have provided a data base for pathogenetic and
CANCER
RESEARCH
prognostic studies and for targeting and monitoring therapeutic
approaches.
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Immunological Characterizations of Patients with Acquired
Immune Deficiency Syndrome, Acquired Immune Deficiency
Syndrome-related Symptom Complex, and a Related Life-Style
Evan M. Hersh, P. W. A. Mansell, J. M. Reuben, et al.
Cancer Res 1984;44:5894-5901.
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